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The rate of DNA replication in a living cell was first measured as the rate of phage T4 DNA elongation in phage-infected ''E. coli''.<sup id="cite_ref-25">[https://en.wikipedia.org/wiki/DNA_replication#cite_note-25 [24]]</sup> During the period of exponential DNA increase at 37 °C, the rate was 749 nucleotides per second. The mutation rate per base pair per replication during phage T4 DNA synthesis is 1.7 per 10<sup>8</sup>.<sup id="cite_ref-26">[https://en.wikipedia.org/wiki/DNA_replication#cite_note-26 [25]]</sup><br/>
=== Transposase === a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the [https://en.wikipedia.org/wiki/Tn3_Transposon Tn3 transposon].<sup id="cite_ref-1">[https://en.wikipedia.org/wiki/Transposase#cite_note-1 [1]]</sup> The existence of transposons was postulated in the late 1940s by [https://en.wikipedia.org/wiki/Barbara_McClintock Barbara McClintock], who was studying the inheritance of [https://en.wikipedia.org/wiki/Maize maize], but the actual molecular basis for transposition was described by later groups. McClintock discovered that some segments of [https://en.wikipedia.org/wiki/Chromosome chromosomes] changed their position, jumping between different loci or from one chromosome to another. The repositioning of these transposons (which coded for color) allowed other genes for pigment to be expressed.<sup id="cite_ref-website1_2-0">[https://en.wikipedia.org/wiki/Transposase#cite_note-website1-2 [2]]</sup> Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause [https://en.wikipedia.org/wiki/Antimicrobial_resistance antibiotic resistance].<sup id="cite_ref-website1_2-1">[https://en.wikipedia.org/wiki/Transposase#cite_note-website1-2 [2]]</sup> Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions.<sup id="cite_ref-MM_3-0">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup> Transposases are classified under [https://en.wikipedia.org/wiki/Enzyme_Commission_number EC number] EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known.<sup id="cite_ref-4">[https://en.wikipedia.org/wiki/Transposase#cite_note-4 [4]]</sup> During the course of human evolution, as much as 40% of the human genome has moved around via methods such as transposition of transposons.<sup id="cite_ref-website1_2-2">[https://en.wikipedia.org/wiki/Transposase#cite_note-website1-2 [2]]</sup><br/> ==== Transposase Tn5 ==== Transposase (Tnp) Tn5 is a member of the [https://en.wikipedia.org/wiki/Ribonuclease RNase] superfamily of proteins which includes retroviral [https://en.wikipedia.org/wiki/Integrase integrases]. Tn5 can be found in ''[https://en.wikipedia.org/wiki/Shewanella Shewanella]'' and ''[https://en.wikipedia.org/wiki/Escherichia Escherichia]'' bacteria.<sup id="cite_ref-Website2_5-0">[https://en.wikipedia.org/wiki/Transposase#cite_note-Website2-5 [5]]</sup> The transposon codes for antibiotic resistance to [https://en.wikipedia.org/wiki/Kanamycin kanamycin] and other aminoglycoside antibiotics.<sup id="cite_ref-MM_3-1">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup><sup id="cite_ref-N1_6-0">[https://en.wikipedia.org/wiki/Transposase#cite_note-N1-6 [6]]</sup> Tn5 and other transposases are notably inactive. Because DNA transposition events are inherently mutagenic, the low activity of transposases is necessary to reduce the risk of causing a fatal mutation in the host, and thus eliminating the [https://en.wikipedia.org/wiki/Transposable_element transposable element]. One of the reasons Tn5 is so unreactive is because the N- and C-termini are located in relatively close proximity to one another and tend to inhibit each other. This was elucidated by the characterization of several mutations which resulted in hyperactive forms of transposases. One such mutation, L372P, is a mutation of amino acid 372 in the Tn5 transposase. This amino acid is generally a leucine residue in the middle of an alpha helix. When this leucine is replaced with a proline residue the alpha helix is broken, introducing a conformational change to the C-terminal domain, separating it from the N-terminal domain enough to promote higher activity of the protein.<sup id="cite_ref-MM_3-2">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup> The transposition of a transposon often needs only three pieces: the transposon, the transposase enzyme, and the target DNA for the insertion of the transposon.<sup id="cite_ref-MM_3-3">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup> This is the case with Tn5, which uses a cut-and-paste mechanism for moving around transposons.<sup id="cite_ref-MM_3-4">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup> Tn5 and most other transposases contain a DDE motif, which is the active site that catalyzes the movement of the transposon. Aspartate-97, aspartate-188, and glutamate-326 make up the active site, which is a triad of acidic residues.<sup id="cite_ref-BC_7-0">[https://en.wikipedia.org/wiki/Transposase#cite_note-BC-7 [7]]</sup> The DDE motif is said to coordinate divalent metal ions, most often magnesium and manganese, which are important in the catalytic reaction.<sup id="cite_ref-BC_7-1">[https://en.wikipedia.org/wiki/Transposase#cite_note-BC-7 [7]]</sup> Because transposase is incredibly inactive, the DDE region is mutated so that the transposase becomes hyperactive and catalyzes the movement of the transposon.<sup id="cite_ref-BC_7-2">[https://en.wikipedia.org/wiki/Transposase#cite_note-BC-7 [7]]</sup> The glutamate is transformed into an aspartate and the two aspartates into glutamates.<sup id="cite_ref-BC_7-3">[https://en.wikipedia.org/wiki/Transposase#cite_note-BC-7 [7]]</sup> Through this mutation, the study of Tn5 becomes possible, but some steps in the catalytic process are lost as a result.<sup id="cite_ref-MM_3-5">[https://en.wikipedia.org/wiki/Transposase#cite_note-MM-3 [3]]</sup> <br/> <br/> <br/> <br/> [https://biolecture.org/Main_Page Main Page] » [https://biolecture.org/UNIST_Geromics_course UNIST Geromics course] » [https://biolecture.org/Geromics_Course_Students_Folder_2024 Geromics Course Students Folder 2024] » [https://biolecture.org/HyoungJinChoi_2024_Geromics_Course HyoungJinChoi 2024 Geromics Course] » [https://biolecture.org/Summary_class_Geromics_2024_HyoungJinChoi Summary class Geromics 2024 HyoungJinCho]